DE69008282T2 - Caffeic acid derivatives, their use for medical treatments and pharmaceutical compositions containing them. - Google Patents

Description

The present invention relates to dihydrocaffeic acid derivatives and their use as medicaments. In particular, the present invention relates to dihydrocaffeic acid derivatives having the ability to induce the production and secretion of nerve growth factor (hereinafter abbreviated as "NGF") in the local tissue of the brain. Furthermore, the present invention relates to prophylactic and therapeutic preparations for regressive disorders of the central nervous system containing these derivatives as active ingredients.

Research into early diagnosis and etiological therapy of various types of age-related diseases has made rapid progress in parallel with the increase in life expectancy of people around the world. Regressive disorders of the central nervous system are therefore also the main subject of research. In particular, senile dementia of the Alzheimer type (hereinafter abbreviated to "SDAT") is becoming a serious social problem, as SDAT is noticeably increasing, especially in developed countries, and is taking a progressive and tragic course.

Especially in recent years, many researchers and doctors have intensively investigated SDAT, but there has been no fundamental clarification of the disease, nor has an effective early diagnosis and therapy been developed.

However, after many pathological observations have been collected, it has been found that direct causes of the memory loss and disorientation that are characteristic early symptoms of SDAT are the progressive degeneration of magnocellular cholinergic pathways and the insufficiency of their responsible region, the aforementioned cholinergic pathways extending from the basal forebrain to the cerebral cortex and hippocampus, the centers for memory and learning. In fact, it has been reported that the symptoms of SDAT have been slightly improved by administering a precursor of acetylcholine biosynthesis or an inhibitor of cholinesterase as an active treatment for the cholinergic neurons of the brain to SDAT patients. However, the results cannot be considered as efficient as expected.

NGF has been the subject of intensive research since its discovery by R. Levi-Montalcini, S. Cohen et al. Some physiological experiments have already clearly demonstrated that NGF is the essential factor for the peripheral nervous system, which affects the differentiation and growth of sensory and sympathetic nerves in a fetus and the survival and maintenance of the functions of the sympathetic neurons in an adult.

NGF is a biologically active substance that is present only in extremely trace amounts, so that despite research over a long period of time, no precise data could be obtained on the distribution and movement of NGF in tissues with regard to direct support of the vital functions of the tissue. Only very recently, a highly sensitive solid phase enzyme immunoassay (hereinafter abbreviated as "ELISA") for identifying the active subunit of NGF, i.e., β-NGF (hereinafter referred to simply as "NGF") has been developed and improved so that the sensitivity in detection and an appropriate specificity for the above-mentioned investigation have been achieved [S. Furukawa et al., J. Neurochem. 40 , 734-744 (1983); S. Korshing and H. Thoenen, Proc. Natl. Acad. Sci. USA 80, 3513- 3516 (1983)].

Furthermore, the NGF gene has been cloned and its structure has been elucidated. A method has also been established to determine the messenger RNA (hereinafter abbreviated as "mRNA") of β-NGF using complementary DNA (hereinafter abbreviated as "cDNA") as a probe [D. L. Shelton and L. F. Reichardt, Proc. Natl. Acad. Sci. USA, 81, 7951-7955 (1984); and R. Heumann et al., EMBO J., 3, 3183-3189 (1984)].

Using this technique, it has been demonstrated that there is a positive correlation between the level of sympathetic nerve supply to the peripheral nervous system and the gene expression of NGF in this nerve-supplied tissue.

More surprisingly, NGF has also been found in the rat central nervous system, particularly in the hippocampus, neocortex, basal forebrain, e.g., the septum, the olfactory bulb, the diagonal band of Broca, and the basal nucleus magnocellularis. It has also become apparent that the level of NGF mRNA is high in the hippocampus and neocortex, while this level is as low in the septum of the basal forebrain as in the other regions of the brain where NGF is not detected [S. Korshing et al., EMBO J., 4, 1389-1393 (1985)]. Subsequently, the results of these experiments have been successively confirmed by other research groups [DL Shelton and LF Reichardt, Proc. Natl. Acad. Sci. USA, 83, 2714-2718 (1986); S. R. Whittemore et al., Proc. Natl. Acad. Sci. USA, 83, 817-821 (1986)].

According to these results, the NGF gene is expressed not only in the peripheral nervous system, but also in the central nervous system. In addition, it has been determined that NGF is produced and secreted in the nerve-supplied regions of the cholinergic pathways that run from the origin of the basal forebrain to the neocortex and hypocampus, i.e. to the centers of memory and learning, and is then taken up at the endings of the nerves and transported retrogradely by axons to the cells (somata) at the origin. It is already considered certain, based on a series of physiological experiments, that NGF is the essential factor for the survival and maintenance of functions in the cholinergic pathways. Thus, these results have shown that NGF also works specifically as a "neurotropic factor" in the central nervous system.

These experiments were subsequently replicated by other research groups and also confirmed by studies concerning NGF receptors and their distribution in the brain.

In the course of research into the function of NGF as a neurotropic factor in the central nervous system, the inventors of the present invention have found that the memory and learning impairments that constitute the early symptoms of SDAT are directly caused by the progressive degeneration of cholinergic pathways and the insufficiency of the responsible region that is triggered thereby. But the real and fundamental reason for the impairments is the insufficiency of NGF production and secretion in specific regions of the brain.

That is, the inventors of the present invention consider a conventional symptomatic therapy against SDAT as a complementary therapy and/or a therapy for improving availability using acetylcholine is not capable of producing remarkably good results. Rather, they believe that more effective results can be achieved if the functional vicious circle between the responsible nerves and the regions under their control can be broken by maintaining the production and secretion of NGF in the cerebral cortex and hippocampus.

In this context, the technology for mass production of human β-NGF has already been achieved by cloning the gene, but the complementary therapy with NGF itself, which is a protein with a molecular weight of more than 10,000, is considerably limited from a pharmacological and pharmaceutical point of view. In particular, the application of NGF to the central nervous system is currently far from being realized.

In view of the foregoing, it is important to search for a low molecular weight compound having the ability to induce the production and secretion of NGF in the specific tissue, thus enabling a substantial and effective supplementary NGF therapy. The present inventors have already reported catechin derivatives having such a function (Ikeda: Japanese Laid-Open Publication No. 63-83020 and Japanese Patent Application No. 63-63516); see also EP-A-0,261,977. In addition, there are also reports by Furukawa et al., [Y. Furukawa et al., J. Biol., Chem., 261 6039 (1986) and FEBS Letters 208, 258 (1986)]. These described compounds are excellent in terms of production and secretion of NGF, but their absorption in case of oral administration and their retention concentration in the blood are not as desired.

An object of the present invention is to provide compounds from which a medicament can be formulated which has the ability to induce the production and secretion of NGF in a specific tissue as an essential and effective adjunct therapy. That is, the invention seeks a compound having the activity of inducing, in areas under the control of specific nerves, the production and secretion of NGF which acts as a "neurotropic factor" for the responsible nerves. Alternatively, a modified compound is sought which is obtained under conventional pharmacological and pharmaceutical considerations. These two types of compound are expected to increase the delivery of NGF to the site of the degenerated nerves and restore their function when the compound is administered in a conventional manner and dosage. In particular, the use of the compounds according to the present invention is particularly effective for the treatment of SDAT with the central nervous system disorders for which a basic therapy has not yet been developed. In the early stage of SDAT symptoms, peripheral administration of the above-mentioned compound can increase the capacity for NGF production and its secretion in the cerebral cortex and hypocampal regions of the central nervous system, thus preventing the progression of the characteristic degeneration in the responsible cholinergic neurons and promoting the repair of the injured neurons and the re-supply of nerves by the surviving neurons. In short, the aim of the present invention is to provide an epoch-making therapy based on a new functional concept making use of the plasticity of the brain.

Another object of the present invention is to provide a medicament which, in case of oral administration, has excellent absorption and good retention concentration in the blood and has the ability to induce the production and secretion of NGF.

The compounds of the present invention are various acyl compounds represented by the general formula (I) wherein the hydroxyl groups of dihydrocaffeic acid are replaced by acyl groups, so that the transfer of the drug into the brain system is increased for these compounds. Therefore, these compounds are considered to be effective drugs for the above-mentioned disorders.

The present inventors have searched for low molecular weight compounds that have the ability to induce the production and secretion of NGF in specific tissues.

As a result, they found that specific derivatives of dihydrocaffeic acid have the function of inducing the production and secretion of NGF, that they are effective for inhibiting the progression and for treating regressive disorders of the central nervous system, and that they can improve the passage of the drug into the brain system. The present invention has been completed on the basis of these discoveries.

The present invention provides dihydrocaffeic acid derivatives represented by the general formula (I)

and salts of these derivatives (wherein R₁ is an alkyl group having 2 or more carbon atoms, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group; where R₂ is

wherein R₃ and R₄ are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an adamantyl group, an aryl group or a substituted aryl group, and wherein X is a direct bond, a methylene group, an oxygen atom or an NH group, provided that R₃ and R₄ are not both simultaneously hydrogen atoms or alkyl groups having three or fewer carbon atoms. The present invention encompasses medications which are effective for inhibiting the progression and for the therapy of regressive disorders of the central nervous system, which medicaments comprise the above-defined derivatives or their salts as active ingredients.

The present invention encompasses the first and second medical uses of these derivatives and their salts.

The present invention is explained in more detail below with reference to the non-limiting description, which is given only as an example.

In the dihydrocaffeic acid derivatives represented by the general formula (I), examples of the alkyl group having two or more carbon atoms for R₁ are straight-chain alkyl groups such as ethyl, propyl, butyl, hexyl, octyl, decyl, lauryl, hexadecyl and stearyl groups, and branched alkyl groups such as isopropyl and isobutyl groups; examples of the aryl group are phenyl group and naphthyl group; examples of the substituted aryl group are benzyl, phenethyl and p-methylphenyl group; examples of the heteroaryl group are pyridyl, pyrimidyl, imidazolyl and furyl group; examples of the substituted heteroaryl group are halogen-substituted pyridyl group, Methylpyridyl group and methylimidazolyl group. In addition, examples of the alkyl group for R₃ and R₄ are the above-mentioned alkyl groups each having two or more carbon atoms and the methyl group; examples of the cycloalkyl group are the cyclopropyl, the cyclobutyl, the cyclopentyl, the cyclohexyl and the cycloheptyl group. In addition, typical examples of the group are

for R2 in the general formula (I) the pyrrolidino, the piperidino, the morpholino and the piperazino group.

Reference is made below to the process for preparing the compound according to the present invention.

The first method of preparation comprises the step of subjecting a compound represented by the general formula (II) and a corresponding acid halide or acid anhydride to an ordinary esterification reaction to obtain a compound represented by the general formula (I),

(wherein R₁ and R₂ are as defined above, and Y is a chlorine or bromine atom).

The above-mentioned esterification reaction is carried out in a solvent such as benzene, toluene, chloroform, tetrahydrofuran or dimethylformamide in the presence of a base such as pyridine, triethylamine or sodium hydroxide at a reaction temperature of 0 to 50°C. However, the compound represented by the general formula (II) can be synthesized by thermal condensation of a commercially available ethyl dihydrocaffeate and a corresponding amine.

Here, "thermal" means that the heating step is carried out at a temperature in the range of room temperature to 200ºC. In most cases, the above reaction proceeds in the absence of any solvent, but in some cases an excess amount of an appropriate amine or an inert solvent such as toluene or xylene may be used.

The second process for preparing the compound according to the present invention comprises the steps of esterifying dihydrocaffeic acid with a corresponding acid halide or acid anhydride, forming a corresponding acid chloride using thionyl chloride and then reacting the acid chloride with a corresponding amine in the presence of a base

(wherein R₁, R₂, R₃, R₄, X and Y are as defined above).

In this case, the above-mentioned base is an organic base such as pyridine or triethylamine, an inorganic base such as sodium hydroxide or potassium hydroxide, or an excess amount of a corresponding amine. The reaction temperature is preferably in the range of 0 to 50°C. The solvent is preferably the above-mentioned organic base, water or an organic solvent such as chloroform, tetrahydrofuran or benzene. Alternatively, the diacyl compound of dihydrocaffeic acid obtained here and a corresponding amine may be subjected to an ordinary condensation reaction using a condensing agent (e.g., dicyclohexylcarbodiimide) as used in an ordinary peptide synthesis.

Next, the following studies were carried out to confirm the efficacy of the compound according to the present invention in inhibiting the progression and treating regressive central nervous system disorders.

That is, NGF was produced and secreted in a culture medium containing the compound of the present invention using a mouse fibroblast cell line, namely, L-M cell line (ATCC, CCLI, 2) described by Furukawa [Y. Furukawa et al., J. Biol. Chem., 261, 6039 (1986)]. The concentration of NGF thus produced was then measured by the highly sensitive ELISA method.

The concentration of NGF was also measured in a system using astroglial cells, which are believed to be the main source of NGF production and secretion in the central nervous system. From the results of these studies, it was clarified that the compounds of the present invention have an extremely good ability to promote the production and secretion of NGF. As a result, it has been confirmed that the compounds of the present invention can be used for prophylactic and therapeutic preparations effective for the treatment of regressive disorders in the central nervous system, particularly for the treatment of SDAT.

In addition, compared to dihydrocaffeic acid, the compounds of the present invention have various acyl groups instead of hydroxyl groups, and therefore have excellent properties in terms of their absorption upon oral administration, their retention concentration in blood and the increase in the concentration of NGF in the brain according to in vivo tests. In addition, they have low acute toxicity. Thus, the compounds of the present invention have to the compounds that possess the hydroxyl groups, many advantages as a medicine.

When a compound of the present invention is used as a prophylactic or therapeutic pharmaceutical for treating regressive central nervous system disorders, the dose and formulation of the compound will, of course, depend on the physical properties of the compound, the symptoms of the patient and also on other factors. In case of oral administration, the dose of the compound for an adult is in the range of 50 to 1000 mg per day, wherein the compound may be administered in a single dose or in multiple doses in the form of tablets, granules, powder, suspension or capsules. In case of non-oral administration, 1 to 100 mg of the compound may be administered in a single dose or in multiple doses in the form of injections, suppositories or isotonic solutions for infusion.

For the preparation of tablets, for example, crystalline cellulose, light anhydrous silica or the like can be used as an adsorbent. As an excipient, corn starch, lactose, calcium phosphate, magnesium stearate or the like can be used. In addition, the compound of the present invention can be used for the preparation of an injection solution in the state of an aqueous solution, an aqueous suspension of cottonseed oil, corn germ oil, peanut oil, olive oil or the like, or an emulsion obtained by using a surfactant such as HCO-60.

The present invention will now be described in more detail with reference to the following examples, which, however, should not be construed as limiting the scope of the invention.

example 1 N-[3-(3,4-Dinicotinoyloxyphenyl)-propionyl]-morpholine

(a) 3 g of dihydrocaffeic acid and 1.43 g of morpholine were dissolved in 20 ml of DMF (dimethylformamide). Then, 202 mg of DNAP (dimethylaminopyridine) and 3.4 g of DCC (dicyclohexylcarbodiimide) were added under cooling. Then, the mixture was allowed to stand overnight. The deposited dicyclohexylurea was removed by filtration and the resulting filtrate was then distilled off under reduced pressure. Then, the residue was washed with ether to obtain 3.8 g of N-[3- (3,4-dihydroxyphenyl)propionyl]morpholine as pale yellow crystals with a melting point of 211 to 213 °C.

(a)' 3 g of ethyl dihydrocaffeate was mixed with 3 g of morpholine, and the mixture was then heated at 150°C for 2 hours with stirring. After cooling, the reaction solution was concentrated and the resulting residue was purified on a silica gel column chromatograph. Elution was then carried out with a mixture of chloroform/methanol (20:1) to obtain 3.7 g of N-[3-[3,4-dihydroxyphenyl)propionyl]morpholine having a melting point of 211 to 213°C.

(b) 2.51 g of N-[3-[3,4-dihydroxyphenyl)propionyl]morpholine obtained in the preceding section (a) or (a)' was suspended in 100 ml of chloroform. Then, 3.56 g of nicotinic acid chloride hydrochloride was added thereto under cooling. Then, 5.6 ml of triethylamine was gradually added to the reaction solution, followed by stirring at room temperature for 4 hours. The reaction solution was washed with a saturated saline solution and then dried over Glauber's salt. The solvent was distilled off under reduced pressure. The resulting residue was purified on a silica gel column chromatograph. Then, elution was carried out with a mixture of chloroform/methanol (30:1) to obtain 1.99 g of N-[3-(3,4-dinicotinolyloxyphenyl)propionyl]morpholine. were received.

Example 2 N-[3-(3,4-dibenzoyloxyphenyl)propionyl]morpholine

(a) 3 g of dihydrocaffeic acid was dissolved in 20 ml of pyridine. Then 5 g of benzoyl chloride was slowly added under cooling. After stirring at room temperature for 2 hours, the solvent was distilled off and the resulting residue was poured into 50 ml of ice water. This solution was neutralized with 6 N hydrochloric acid and then extracted twice with 50 ml of chloroform. After washing with a saturated saline solution, the extracted material was dried over Glauber's salt. Then the solvent was distilled off under reduced pressure to obtain 6.8 g of 3-(3,4-dibenzoyloxyphenyl)propionic acid.

(b) 6 g of 3-(3,4-dibenzoyloxyphenyl)propionic acid was dissolved in 20 ml of benzene, and 2 g of thionyl chloride was added. After stirring at 60°C for 2 hours, the solvent was distilled off to obtain crude 3-(3,4-dibenzoyloxyphenyl)propionic acid chloride. To 50 ml of chloroform was added 2.8 g of morpholine, and then the above acid chloride was slowly added dropwise under cooling. After stirring at room temperature for 2 hours, the reaction solution was washed with a saturated saline solution and then dried over Glauber's salt. Then, the solvent was distilled off under reduced pressure. The resulting residue was purified on a silica gel column chromatograph. After elution with ethyl acetate, 6.7 g of N-[3-(3,4-dibenzoyloxyphenyl)propionyl]morpholine were obtained.

Examples 3 to 20

The same reactions and treatments as in Examples 1 or 2 were carried out to obtain the compounds of Examples 3 to 20 as shown in Table 1. Table 1 (I) Compound of the present invention Structure example oily Table 1 (II) Structure example oily Table 1 (III) Structure Example amorphous powder oily Table 1 (IV) Example (pure) Table 1 (V) Example (pure) Table 1 (VI) Example (pure) Table 1 (VII) Example (pure)

Example 21 [The function promoting the production and secretion of NGF in L-M cells of a mouse]

This experiment was carried out according to the procedure of Furukawa et al. [Y. Furukawa et al., J. Biol. Chem., 261, 6039 - 6047 (1986)].

That is, L-M cells were pre-cultured in the culture medium 199 (manufactured by Gibco Co., Ltd.) containing 0.5% peptone. Then, the cells were transferred to a 24-well culture plate (manufactured by Falcon Co., Ltd., the area for culture per well is 2.1 cm2) so that there were about 3 x 104 cells/well. Then, the cells were cultured at 37°C for 3 days so that they gave a completely confluent lawn (about 106 cells/well). The culture medium was then replaced with the culture medium 199 (0.5 ml/well) containing 0.5% bovine serum albumin (the 5th section, manufactured by Armour Co., Ltd.). The compound to be tested was contained in this culture medium at a predetermined concentration, and after 24 hours, the concentration of NGF in the culture medium was measured by the highly sensitive ELISA method [S. Furukawa et al., J. Neurochem., 40, 734-744 (1983)].

The results showed an increase in the concentration of NGF in the culture media containing the compound under test compared to controls without this compound. The detection limit of the ELISA method was 0.25 pg/ml and the NGF concentration of the control was usually from 50 to 200 pg/0.5 ml of well fluid. Each value of the results is an average of 4 measurements using the same preparation of cells. The results are presented in Table 2.

Example 22 [The function promoting the production and secretion of NGF in astroglial cells from the brain of a mouse]

Astroglial cells were isolated from the forebrain of a mouse and then transferred into a culture medium [S. Furukawa et al., Biochem. Biophys. Res. Commun., 136, 57-63 (1986)].

That is, the brain of an 8-day-old mouse was cut into thin slices and then washed with phosphate buffered physiological saline (hereinafter abbreviated as "PBS") containing neither calcium nor magnesium. The brain sections were then treated in PBS containing 0.25% trypsin at 37ºC for 30 minutes. The brain tissue was then sufficiently loosened by a Pasteur pipette to form a suspension. The latter was then centrifuged at 200 x g for 5 minutes to recover the cells and cell aggregates. They were resuspended in Dulbecco's modified Eagle's culture medium (hereinafter abbreviated as "DMEM culture medium", manufactured by Gibco Co., Ltd.) containing 10% fetal bovine serum, 5 x 10-5 mM sodium chloride, and 10% fetal bovine serum. units/ml penicillin and 5 ug/ml streptomycin. Primary culture was carried out for a period of 10 to 14 days, with the same type of culture medium being renewed every 3 days. After the cells reached a confluent state, they were treated with trypsin, then they were divided and re-seeded in another culture medium. The seeding was repeated 2 or more times to obtain a morphologically uniform cell population. In this experiment, the PAP staining method (peroxidase-antiperoxidase staining method) is used, which makes use of a rabbit antiserum directed against human glial fibrillary protein (GFAP), and the above-mentioned cell population was stained by 97% or more in this way. This cell population would be called astroglial cells.

The astroglia cells were seeded in an amount of approximately 3 x 10⁴ cells/well into a 24-well culture plate (prepared from Falcon Co., Ltd., the area of one well is 2.1 cm2). Then, these cells were cultured for 3 days in a DMEM culture medium containing 10% fetal bovine serum so that the cell layer was confluent (about 107 cells/well). The culture medium was replaced with a DMEM culture medium containing 0.5% bovine serum albumin (the fifth section) (0.5 ml/well), and then the astroglial cells were cultured for 3 days. This culture medium was also renewed every 3 days to bring the cells into a resting state. Then, the culture medium was replaced with 0.5 ml of a similar culture medium containing a compound to be tested at a predetermined concentration. After 24 hours, the concentration of NGF in the culture medium was measured by the highly sensitive ELISA method. The results showed an increase in the concentration of NGF in the culture media containing the compound under test compared to controls without this compound. The detection limit of the ELISA method was 0.25 pg/ml and the NGF concentration of the control was generally from 1 to 10 pg/0.5 ml of well fluid. Each value of the results is an average of 4 measurements using the same preparation of cells. The results are presented in Table 3. Table 2 (The function of promoting the production and secretion of NGF in mouse LM cells) Compound (Example No.) Sample concentration NGF concentration Increase in NGF (Increase over control) Control Table 3 (The function of promoting the production and secretion of NGF in mouse brain astroglial cells) Compound (Example No.) Sample concentration NGF concentration Increase in NGF (increase over control) Control

Claims (11)

1. Dihydrocaffeic acid derivative represented by the general formula (I) (wherein R₁ is an alkyl group having 2 or more carbon atoms, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group; where R₂ is wherein R₃ and R₄ are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an adamantyl group, an aryl group or a substituted aryl group, and wherein X is a direct bond, a methylene group, an oxygen atom or an NH group, but wherein R₃ and R₄ are not simultaneously hydrogen atoms or alkyl groups with three or fewer carbon atoms), and salts of this derivative. 2. The compound according to claim 1, wherein R₁ is a phenyl group, a pyridyl group, a t-butyl group or a propyl group. 3. The compound of claim 1 or 2, wherein R₂ is is. 4. The compound of claim 1 or 2, wherein R₂ is is. 5. The compound of claim 1 or 2, wherein R₂ is. 6. The compound of claim 1 or 2, wherein R₂ is. 7. The compound of claim 1 or 2, wherein R₂ is. 8. Medicament for preventing the progression and for the therapy of regressive disorders of the central nervous system, which contains a dihydrocaffeic acid derivative represented by the general formula (I) and its salt as active ingredients. 9. The compound of any one of claims 1 to 7 for use in inducing the production and secretion of nerve growth factor in brain tissue. 10. The compound of any one of claims 1 to 7 for use as a prophylactic or medicament against regressive disorders of the central nervous system. 11. Use of a compound according to any one of claims 1 to 7 for the manufacture of a medicament for the prophylaxis or therapy of regressive disorders of the central nervous system.